PROJECT SUMMARY Ocular surface neoplasms such as conjunctival melanoma and ocular surface squamous neoplasia are potentially blinding or fatal lesions of the eye that arise from the conjunctiva or corneal limbus. Early detection and treatment is important for successful outcomes, and treating high-risk pre-malignant lesions before they become malignant is favorable. However, establishing a clinical diagnosis is often not straightforward due to overlap in the appearance of different lesion types. Furthermore, a clinically benign lesion such as primary acquired melanosis (PAM) can potentially harbor microscopic cellular atypia, which confers a significant risk of conversion to melanoma. The only currently available method to reliably distinguish benign from pre-malignant lesions is to excise the lesion for biopsy, which results in many benign lesions being removed in order not to miss any malignant ones. Topical chemotherapeutic options exist, but their appropriate use depends upon accurate diagnosis and monitoring of the condition, which underscores the importance of a reliable and sensitive non-invasive imaging tool to detect ocular surface malignancy. To address this need, we propose a new optical coherence tomography (OCT) modality, dual-band dual-scan inverse spectroscopic OCT (D2ISOCT), to provide sensitive and comprehensive quantification of both microangiography, microvascular oxygenation and nanoscale ultra-structural properties. No other existing technology enables such measurements. Previous studies have detected increased oxygen saturation and ultrastructural alterations in pre-malignant tissue. We have also conducted an ex vivo study from human conjunctival biopsies, showing that increases in an ISOCT marker of tissue ultra-structure alteration, as well as changes in metabolic markers detected by two photon intrinsic autofluorescence microscopy were correlated with ocular surface malignancy. We then hypothesize that ISOCT ultra-structural markers, microvascular density and sO2 are sensitive markers for the detection of ocular surface malignancy and pre-malignant states. We have two specific aims. 1) We will develop a novel dual-band dual-scan ISOCT (D2ISOCT) system for in vivo analysis of the human ocular surface. 2) We will conduct a pilot clinical study to validate the D2ISOCT system for detecting malignant and pre-malignant changes in ocular surface lesions in human subjects. IMPACT ON PUBLIC HEALTH: 1) The success of this project will lead to a groundbreaking new imaging device for the evaluation of lesions of the ocular surface. The technique may help to guide appropriate treatment for patients and improve their outcomes. 2) The validity of the imaging method could lead to further clinical study on predictive imaging markers for other malignant lesions of the eye, including iris and retinal tumors.